Villin, an epithelial cell microvillar protein bundles, nucleates, caps, or severs actin filaments in response to changes in the concentration of calcium, phosphatidylinositol 4,5-bisphosphate, and tyrosine phosphorylation, which are often immediate consequences of cell activation. Our previous studies have identified that villin's ligand-binding properties are mechanistically important to its role in cell migration. In this proposed continued study, we will examine the biochemical and molecular mechanisms by which tyrosine phosphorylation of villin regulates cell migration. The specific goals of the proposed research are: (1) To understand at the molecular level the relationship between the structure and function of tyrosine phosphorylated villin. (2) To identify and characterize the tyrosine kinase(s) that regulate(s) villin phosphorylation and villin-induced cell migration. (3) To identify and characterize the tyrosine phosphatase(s) that regulate(s) villin dephosphorylation and villin-induced cell migration. Ultimately, we strive by this study to understand two critical functions: first, to understand how tyrosine phosphorylation of villin and its ligand-binding properties regulate epithelial cell signal transduction and motility; second how actin-binding proteins interact with other second messengers to regulate cell structure and function. We will characterize tyrosine phosphorylation of villin and its ligand-binding properties using the following approaches: in vitro reconstitution using recombinant villin proteins; the intestinal epithelial cell line, Caco-2; over expression of villin and enzymes, that regulate tyrosine phosphorylation of villin, in the Tet-Off inducible cell lines, HeLa or MDCK; and develop primary cultures of enterocytes from villin knock-out mice infected (using adenoviral vectors) with wild-type and phosphorylation site mutants of villin. These studies have the potential to allow modification of motility for enhancement of normal physiology and for amelioration of disease. An inhibition of epithelial cell motility can be significant in several diseases, including inflammatory bowel disease, celiac disease, and colon cancer.